CY-8090 Human TXNIP ELISA Kit
Contents
1. ne Human TXNIP ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human TXNIP SN CircuLex Human TXNIP ELISA Kit N Cat CY 8090 C Intended Nr T 1 AOI aT EEEE 1 Lor rge ratore Toy ERN HM 2 Qy Principle of the Assay 2 3 Materials Provided iecoris 3 Materials Required but not Provided 4 Precautions and Recommendations 5 Sample Collection and Storage 6 Detailed Protoeoluiieeseur ca ees rina ux eta pocs 7 8 rior 9 Measurement Range esee 9 Troubleshooting u eere ren ao erret tont 10 Reagent Stability usi cessere ttis 10 Sample Preparation ceci rere epo naus 11 Assay Characteristics cccssssscecscacsecssesecessnceas Example of Test Results Referentes avete tu ERE IRUDUURN ArtR vida ANGUS 20 Intended Use The CycLex Research Product Circ an TXNIP ELISA Kit is used for the quantitative measurement of human TXNIP in cell lysate culture medium and serum This assay kit is for research use o d not for use in diagnostic or therapeutic procedures Storage Upon receipt store all con at 4 C Don t expose reagents to ex e light C CY 8090 1 Version 150601 Human TXNIP ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Thioredoxin interacting protein TXNIP2. Buffer The concentration of the TXNIP in vial should be 200 ng mL which is referred as a 10X Master Standard of TXNIP Prepare Standard Solutions as follows Use the 10X Master Standard to produce a dilution sefies below Mix each tube thoroughly before the next transfer The 1 200 pg mL standard Std 1 Serves as the highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Master Standard 100 uL of 10X Master Standard 200 ng mL 900 uL 20 ng mL Std 1 60 uL of Master Standard 20 ng mD 940 uL 1 200 pg mL Std 2 300 uL of Std 1 1 200 pg ml 300 uL 600 pg mL Std 3 300 uL of Std 2 600 pg nil 300 uL 300 pg mL Std 4 300 uL of Std 3 300 pg ml 300 uL 150 pg mL Std 5 300 uL of Std 4 150 pg ml 300 uL 75 pg mL Std 6 300 uL of Std 5 75 pg ml 300 uL 37 5 pg mL Std 7 300 uL of Std 6 87 5 pg ml 300 uL 18 8 pg mL Blank 300 uL 0 pg mL Note Do not use a Repeating pipette Change tips for every dilution Wet tip with Dilution Buffer before dispensing Unused portions of Master Standard should be aliquoted and stored at below 70 C immediately Avoid multiple freeze and thaw cycles Sample Preparation Serum and plasma samples require a 50 to 100 fold dilution Othefibiological samples and cell lysate require neat to appropriate dilution Cat CY 8090 T Version 150601 Human TXNIP ELISA Kit L ircuLe
3. coated plate Coat wells of a 96 well plate with 100 uL well of 25 ug mL polyazelysin in PBS for 4 12 hours at 37 C Subsequently wash the wells three times with PBS C Treatment of cells 1 Plate adherent cells in the PLL coated 96 well plate at around 4 x 10 cells well 2 Incubate the plate at 37 C for 7 12 hours in CO incubator 3 Add an appropriate amount of a test compound or glucose and a vehicle for a test compound to each well 5 Incubate the plate at 37 C for appropriate time in CO incubator D Cell extraction Note This protocol has been successfully applied to HeLa cell line Users should optimize the cell extraction procedure for their own applications 1 Wash cells three times with iee cold PBS Remove any remaining PBS by decanting Invert the plate and blot it against clean paper towels At this point the cells in the plate can be frozen at below 70 C and lysed at a later date 2 Lyse the cells by adding 0 mL of the cell lysis buffer for 60 90 minutes at 4 C with rotating at ca 300 rpm by an orbital microplate shaker To get a rough tdea you could adjust the cell concentration to around 1 2 x 10 cells mL in the cell lysis buffer Resulting protein concentration of the HeLa cell lysate should be 0 3 0 4 mg mL using this procedure The appropriate volume of the cell lysis buffer depends on the cell line the cell number and the amount Ofttotal TXNIP For example 1 2 x 10 HeLa cells can be l
4. antibody specific for human TXNIP is added to the wells Following a wash to remove any unbound antibody HRP conjugate the remaining conjugate is allowed fo react with the substrate H202 tetramethylbenzidine The reaction is stopped bysaddition of acidic solution and absorbance of the resulting yellow product is measured at 450 nm The absorbance is proportional to the concentration of human TXNIP A standard curve is constructed by plotting absorbance values versus human TXNIP concentrations of calibrators and concentrations of unknown samples are determined using this standard curve Cat CY 8090 2 Version 150601 TM Human TXNIP ELISA Kit 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure Add 100 uL of diluted samples to the wells 1 Incubate for 1 5 hours at room temp Wash the wells O Add 100 uL of HRP conjugated anti human TXNIP antibody 1 Incubate for 1 5 hours at room temp Wash the wells Add 100 uL of Substrate Reagent Add 100 uL of Stop Solution Q t Measure absorbance at 450 nm Materials Provided All samples and standards should be assayed in e The following components are supplied and are sufficient for the one 96 well microplate kit Microplate One microplate supplied ready to with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated human TXNIP antibody as a capture antibody 10X Wash Buf
5. at 450 nm Cat CY 8090 8 Versions 150601 Th Human TXNIP ELISA Kit L 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Calculations Average the duplicate readings for each Standard Solution control and sample and subtract the average zero standard optical density Plot the optical density for the standards versus the concentration of the standards and draw the best curve The data can be linearized by using log log paper and regression analysis may be applied to the log transformation To determine the human EXNIP concentration of each sample first find the absorbance value on the y axis and extend a horizontal line to the standard curve At the point of intersection extend a vertical line to the x axis and read the corresponding human TXNIP concentration If the samples have been diluted the concentration read from the standard curve must be multiplied by the dilution factor 1 The dose response curve of this assay fits best to a sigmoidal four parameter logistic jequation The results of unknown samples can be calculated with any computer program haying aifour parameter logistic function It is important to make an appropriate mathematical adjustimenft to accommodate for the dilution factor 2 Most microtiter plate readers perform automatic calculations of analyte concentration The calibration curve is constructed by plotting the absorbance Y of calibrators Versus log of the known concen
6. ed at below 70 C for extended periods of time Avoid repeated freeze thaw cycles Note Citrate plasma has not been validated for use in this assay Other biological samples Remove any particulates by centrifug ssay immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cyeles C CY 8090 6 Versions 150601 Thi Human TXNIP ELISA Kit L 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex Human TXNIP ELISA Kit is provided with removable strips of wells so the assay can be carried out on separate occasions using only the number of strips required for the particular determination Since experimental conditions may vary an aliquot of the human TXNIP Standard within the kit should be included in each assay as a calibratot Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay r agents are supplied ready to use with the exception of 10X Wash Buffer and Human TXNIP Standard 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer to 900 mL of deionized distilled water Mix well Store at 4 C for two weeks or 20 C for long term storage 2 Reconstitute Human TXNIP Standard with 1 0 mL of Dilution
7. eionized water of the highest quality Disposable paper towels C CY 8090 4 Version 150601 Human TXNIP ELISA Kit TM L 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations Allow all the components to come to room temperature before use All microplate strips that are not immediately required should be returned to the zip lock pouch which must be carefully resealed to avoid moisture absorption Do not use kit components beyond the indicated kit expiration date Use only the microtiter wells provided with the kit Rinse all detergent residues from glassware Use deionized water of the highest quality Do not mix reagents from different kits The buffers and reagents in this kit may contain preservatives or other chemicals Care should be taken to avoid direct contact with these reagents Do not mouth pipette or ingest any of the reagents Do not smoke eat or drink when performing the assay or in areas where samples or reagents are handled e Dispose of tetra methylbenzidine TMB containifig solutions in compliance with local regulations Avoid contact with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide Wear gloves and eye protection when handling immunodiagnostic materials and samples of human origin and these reagents In case of contact witli the Stop Solution and the Substrate Solution wash
8. fer One bottle containin of 10X buffer containing Tween 20 Dilution Buffer One bottle contai Standard and sample dilution Rea of 1X buffer use for reconstitution of Human TXNIP aining 200 ng of lyophilized recombinant human TXNIP Human TXNIP Standard Ci HRP conjugated Detection A y One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti human T antibody Ready to use Substrate Reag tle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready t o Stop Solutio e containing 20 mL of 1 N H2SO Ready to use C CY 8090 3 Versions 150601 Human TXNIP ELISA Kit CircuLex User s Mani For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Precision repeating pipettor Orbital microplate shaker Microcentrifuge and tubes for sample preparation Vortex mixer s of 450 540 nm e read at a single Plate reader capable of measuring absorbance in 96 well plates at dual wa Microplate washer optional Manual washing is possible but not preferable Dual wavelengths of 450 550 or 450 595 nm can also be used The O b wavelength of 450 nm which will give a somewhat higher reading Software package facilitating data generation and analysis opti 500 or 1000 mL graduated cylinder Reagent reservoirs D
9. m temperature ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The incubation time may be extended up to 30 minutes if the reaction temperature is below than 20 C 11 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 12 Measure absorbance in each well using aspectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a single wayelength can be used Wells must be read within 30 minutes of adding the Stop Solution Note 1 Complete removal of liquid at each step is essential to good performance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against Clean pap r towels Note 2 Reliable standard curves are obtained when either O D values do not exceed 0 25 units for the blank Zero concentration or 3 0 units for the highest standard concentration The plate shouldbe monitored at 5 minute intervals for approximately 30 minutes Note 3 1f the miicfoplate reader is not capable of reading absorbance greater than the absorbance of the highestystandard perform a second reading at 405 nm A new standard curve constructed sing the values measured at 405 nm is used to determine TXNIP concentration of off scale Samples The readings at 405 nm should not replace the on scale readings
10. ncer Res 63 432 440 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info 2 cyclex co jp URL http www cyclex o jp CycLex CircuLex products are supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products without prior written approval from CycLex Co Ltd To inquire about licensing for such commercial use please contact us via email Cat CY 8090 20 Version 150601
11. oduct CircuLex Human TXNIP ELISA Kit have been tested for stability Reagents should not be used beyondgthe stated expiration date Upon receipt kit reagents should be stored at 4 C except the reconstituted XNIP Standard must be stored at below 70 C The Microplate should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack Cat CY 8090 10 Version 150601 Human TXNIP ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Preparation Several extraction methods can be used for measurement cellular total TXNIP The following protocol has been shown to work with a number of different cell lines and is provided as an example of suitable methods It is strongly advised that the user always performs an initial experiment to determine the proper dilution to be used in subsequent experiments This needs not be any more than single time point assay using serial dilutions of the cell lysate One eight well strip of the substrate plate should be sufficient for this initial experiment All steps of cell lysate preparation should be performed at 4 C and recovered cell lysates should be kept at below 70 C Preparation of Cell Lysate A Preparation of cell lysis buffer 20 mM Tris HCl pH 7 5 250 mM NaCl 10 glycerol 0 1 NP 40 5 mM4EDTA 1 mM EGTA 0 2 mM PMSF 1 ug mL pepstatin 0 5 ug mL leupeptin 0 2 mM DTT B Preparation of poly L lysine PLL
12. res Fig 7 Human TXNIP levels in 40 sera of healthy volunteers Human TXNIP concentration n 2 40 12 J Q aa Q E et amp S e S a 6r Z r E g e E un O e J 1 3 0 C CY 8090 19 Version 150601 Human TXNIP ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures References Wang Y De Keulenaer G W and Lee R T 2002 J Biol Chem 277 26496 26500 la Schulze P C De Keulenaer G W Yoshioka J Kassik K A and Lee R T 2002 Circ Res 91 689 695 3 Nishiyama A Matsui M Iwata S Hirota K Masutani H Nakamura H Takagi a Sono H Gon Y and Yodoi J 1999 J Biol Chem 274 21645 21650 4 Junn E Han S H Im J Y Yang Y Cho E W Um H D Kim D K Lee K W Han P L Rhee S G and Choi I 2000 J Immunol 164 6287 6295 n Zhou R Tardivel A Thorens B Choi I Tschopp J 2010 Nat Immunol 11 3640 6 Parikh H Carlsson E Chutkow WA et al 2007 PLoS Med 4 e158 1 Stoltzman CA Peterson CW Breen KT Muoio DM Billin AN Ayer DE 2008 Proc Natl Acad Sci USA 105 6912 6917 8 Fidler IJ Radinsky R 1996 J Natl Cancer Inst 88 1700 1708 9 Nakamura H Masutani H Yodoi J 2006 Semin Cancer Bioh16 444 45 1 10 Goldberg SF Miele ME Hatta N Takata M Paquette Straub C Freedman LP et al 2003 Ca
13. search Use Only Not for use in diagnostic procedures 3 Linearity To assess the linearity of the assay samples containing and or spiked with high concentrations human TXNIP were serially diluted with Dilution Buffer to produce samples with values within the dynamic range of the assay Linearity TXNIP ng mg total protein 0 1 2 3 4 Sample Dilution Ratio 4 C CY 8090 15 Version 150601 Human TXNIP ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Glucose dose dependent production of TXNIP in HeLa cells after glucose treatment for 24 hours cultured in 24 well plate TXNIP level in HeLa cell lysate after Glucose treatment for 24hrs TXNIP conc ng mg total protein 0 5 10 15 20 25 Glucose conc mM Fig 2 NaN3 dose dependent inhibition of TXNIP produetion in HeLa cells after NaN3 treatment for 24 hours cultured in 24 well plate TXNIP level in HeLa cell lysate after NaN treatment for 24hrs gt E d zi Ez g 3 E on E ob 3 I Ei 5 Z E 0 02 0 04 0 06 0 08 0 10 NaN conc 496 Cat CY 8090 16 Version 150601 Human TXNIP ELISA Kit CircuLex User s Manil For Research Use Only Not for use in diagnostic procedures Fig 3 Glucose dose dependent production of TXNIP in HeLa cells after glucose treatment for 24 hours cult
14. skin thoroughly with water and seek medical attention when necessary Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly CAUTION Sulfuric Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 8090 5 Version 150601 Human TXNIP ELISA Kit CircuLex User s Mani For Research Use Only Not for use in diagnostic procedures Sample Collection and Storage Cell lysates Prepare cell lysates Assay immediately or store the samples on ice for a few hours b assaying Aliquots of the samples may also be stored at below 70 C for extended periods of tim Avoid repeated freeze thaw cycles Serum Use a serum separator tube and allow samples to clot for 60 30 minutes samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately or sto ice for up to 6 hours before assaying Aliquots of serum may also be stored at below 70 C fo periods of time Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA Naz as the anticoagulant If possible colle e plasma into a mixture of EDTA Naz and Futhan5 to stabilize the sample against spontane in complement activation Immediately centrifuge samples at 4 C for 15 minutes at 1 000 x immediately or store samples on ice for up to 6 hours before assaying Aliquots of plasma ma e stor
15. the endogenous inhibitor of thioredoxin also knowngas vitamin D3 up regulated protein 1 1 2 or thioredoxin binding protein 2 3 inhibits thioredoxin antioxidative function by binding to its active site thiols 3 4 It was also demonstrated that TXNIP plays a crucial role for caspase 1 activation caused by high glucose treatment in murine p eells by direct interaction with the NLRP3 inflammasome 5 TXNIP dependent inflammasome activation appears to be specific for NLRP3 as TXNIP deficiency did not affect the activity of other inflammasomes e g NLRC4 and AIM 5 TXNIP levels are elevated in subjects with type 2 diabetes mellitus 6 and its expression is induced by glucose 6 phosphate through an intracellular transcriptional complex of MondoA and Max like protein X 7 TXNIP is induced by various types of cellular stress including oxidative stress UV irradiation heat shock and apoptotic signaling 8 and is often suppressed in various human tumors 9 10 Overexpression of TXNIP inhibits proliferation via cell cycle arrest and pr moftes apoptosis 1 4 Principle of the Assay The CycLex Research Product CircuLex Human TXNIP ELISA Kit employs the quantitative sandwich enzyme immunoassay technique An antibody specific fomhuman TXNIP is pre coated onto a microplate Standards and samples are pipetted into the wells and the immobilized antibody binds any human TXNIP present After washing away any unbound substances an HRP conjugated
16. tration X of calibrators using the four parameter function Alternatively the logit log function can be used to linearize the calibration curve i e logit of absorbance Y is plotted versus log of the known concentration X of calibrators Measurement Range The measurement range is 1 200 pg mL to 18 8 pg mb Any sample reading higher than the highest standard should be diluted with Dilution Buffer in higher dilution and re assayed Dilution factors need to be taken into consideration in calculating the human TXNIP concentration Cat CY 8090 9 Versions 150601 Human TXNIP ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting j The Standard Solutions should be run in duplicate using the protocol described in the Detailed Protocol Incubation times or temperatures significantly different from those specified may give erroneous results 2 Poor duplicates accompanied by elevated values for wells containing no sample indicate insufficient washing If all instructions in the Detailed Protocol were followed accurately such results indicate a need for washer maintenance 3 Overall low signal may indicate that desiccation of the plate has occurred betweeitthe final wash and addition of Substrate Reagent Do not allow the plate to dry out Add Substrate Reagerit immediately after wash Reagent Stability All of the reagents included in the CycLex Research Pr
17. uman TXNIP giving absorbance than mean absorbance of blank plus three standard deviations of the absorbance of blank A b 3SD blank is calculated to be better than 9 41 pg mL of sample based on eighteen assays Dilution Buffer is pipetted into blank wells Typical Standard Curve TXNIP Standard Curve A450 0 200 400 600 800 1 000 1 200 TXNIP pg ml C CY 8090 13 Versions 150601 Human TXNIP ELISA Kit CircuLex User s Manil For Research Use Only Not for use in diagnostic procedures 2 Precision Intra assay Precision Precision within an assay Three samples of known concentration were tested seven times on one plate to assess intra precision Intra assay Within Run n 7 CV 3 5 8 8 96 Human TXNIP concentration ng mg total protein No Sample 1 Sample2 Sample 3 1 1 1 3 1 4 9 2 1 2 3 1 4 4 3 1 2 3 2 4 8 4 1 3 3 1 4 8 5 1 1 2 9 4 9 6 1 0 3 0 4 7 7 1 0 3 3 4 Inter assay Precision Precision between assays Three samples of known concentration w sted in three separate assays to assess inter assay precision e nter assay Run to Run n 3 CVz1 6 4 3 Human T ntration ng mg total protein ample 1 Sample 2 Sample 3 1 2 3 1 5 7 1 2 3 1 5 8 1 3 3 0 5 3 ax 1 3 3 1 5 8 min 1 2 3 0 5 3 mean 1 2 3 1 5 6 SD 0 0 0 0 0 2 CV 3 4 1 6 4 3 C CY 8090 14 Version 150601 Human TXNIP ELISA Kit CircuLex User s Manil For Re
18. ured in 96 well plate TXNIP level in HeLa cell lysate after Glucose treatment for 24hrs oO 2 TXNIP conc ng mg total protein 0 5 10 15 20 25 Glucose conc mM 7V Fig 4 NaN3 dose dependent inhibition of TXNIP ion in HeLa cells after NaN3 treatment for 24 hours cultured in 96 well plate TXNIP level in HeLa cell lysate after NaN treatment for 24hrs y T TXNIP conc ng mg total protein w 0 00 0 02 0 04 0 06 0 08 0 10 NaN conc C CY 8090 17 Version 150601 Human TXNIP ELISA Kit CircuLex User s Manil For Research Use Only Not for use in diagnostic procedures Fig 5 Concentrations of human TXNIP in HeLa cell lysate after addition of 25 mM glucose at indic times cultured in 96 well plate Time course of TXNIP productin in HeLa cell lysate after 25mM Glucose treatment Q 0 1 2 4 6 8 9 24 Time course hr ww Fig 6 Concentrations of human TXNIP in HeLa c e after depletion of glucose at indicated times cultured in 96 well plate T TXNIP conc ng mg total protein t2 Time course of TXNIP productin in HeLa cell lysate after 0 01 NaN treatment 3L 2 1L 0 0 1 2 4 6 8 9 24 Time course hr f TXNIP conc ng mg total protein C CY 8090 18 Version 150601 Human TXNIP ELISA Kit CircuLex User s Manil For Research Use Only Not for use in diagnostic procedu
19. x User s Manual For Research Use Only Not for use in diagnostic procedures Standard Assay Procedure for Human TXNIP j Remove the appropriate number of microtiter wells from the foil pouch and place them into the well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C 2 Dilute serum sample with Dilution Buffer See Sample Preparation above o2 Pipette 100 uL of Standard Solutions Std1 Std7 Blank and the diluted samples in duplicates into the appropriate wells 4 Incubate the wells at room temperature ca 25 C for 1 5 hours shaking at a 9300 rpm on an orbital microplate shaker Nn Wash 4 times by filling each well with Wash Buffer 350 uL using a sq irt4bottle multi channel pipette manifold dispenser or microplate washer 6 Add 100 uL of HRP conjugated Detection Antibody into each well N Incubate the wells at room temperature ca 25 C for 1 5 hours shaking at ca 300 rpm on an orbital microplate shaker oo Wash 4 times by filling each well with Wash Buffer 350 tL using a squirt bottle multi channel pipette manifold dispenser or microplate washer 9 Add 100 uL of Substrate Reagent to each well Avoid exposing the microtiter plate to direct sunlight Covering the wells with e g aluminum foil i recommended Return Substrate Reagent to 4 C immediately after the necessary volume is removed 10 Incubate the wells at roo
20. ysed in 0 1 mL of the cell lysis buffer Cat CY 8090 11 Version 150601 Human TXNIP ELISA Kit CircuLex User s Manil For Research Use Only Not for use in diagnostic procedures 3 Centrifuge at 3 500 rpm for 15 minutes at 4 C using a microplate bucket Or transfer the cell lysa to microcentrifuge tubes and centrifuge at 15 000 rpm for 5 minutes at 4 C 4 Transfer the clear cell lysates to a new 96 well plate or clean microcentrifuge tubes Dilute th el lysates 10 times with Dilution Buffer 100 uL of these diluted cell lysates are ready fo the section Standard Assay Procedure for Human TXNIP at page 8 Typical data using this protocol are shown in Fig 3 and Fig 6 page 17 and 18 The cell lysates can be stored at below 70 C Avoid multiple freeze thaw cycles thaw the cell lysates centrifuge at 15 000 rpm for 5 minutes at 4 C again since the cell lysates s clear of any sediments or particulate matter NOTE THE ABOVE PROCEDURES ARE INTENDED ONLY AS ELINE THE OPTIMAL EXPERIMENTAL CONDITIONS WILL VAR DING ON THE PARAMETERS BEING INVESTIGATED AND MUST ERMINED BY THE INDIVIDUAL USER NO WARRANTY OR GUARANTEE OF PERFORMANCE USING THESE PROCEDURES IS MADE OR S C CY 8090 12 Version 150601 Human TXNIP ELISA Kit L ircuLex User s Manual AN For Research Use Only Not for use in diagnostic procedures Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of h
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